image of CyQuant Direct Assay Kit and PrestoBlue reagent bottle

Two-parameter cell viability assay

The PrestoBlue and CyQUANT Direct confirmation assay uses orthogonal assay mechanisms to provide a more stringent measure of viability than any single assay parameter. PrestoBlue reagent responds to the reducing environment of viable cells and the CyQUANT Direct assay reports on the number of live cells with intact cell membranes.

The combined assay workflow is optimized for automation in 96-well or 384-well microplates using a bottom-read fluorescence plate reader.

This protocol can be used for:

  • Quantifying live cells using a fluorescence microplate reader

You will need the following for this protocol:

Assay protocol

1. Pre-incubate or dispense cells in appropriate microplate wells
2. Add PrestoBlue 10X reagent to microplate wells (see recommended volumes)
3. Incubate at 37°C for 10 minutes
4. Read fluorescence at 590 nm
5. Add CyQUANT Direct 2X detection reagent to microplate wells (see instructions for making 2X detection reagent and recommended well volumes)
6. Incubate at 37°C for 60 minutes
7. Read fluorescence at 538 nm
7. Plot a curve of relative fluorescence units vs. drug concentration to generate quantitative results for both assays
Spectral information and storage
  PrestoBlue 10X Cell Viability Reagent CyQUANT Direct 2X detection reagent
Excitation/Emission (in nm) 560/590 485/538
Storage conditions 4°C 4°C


Protocol tips

  • The assay can tolerate a variety of cell culture media components including phenol red and up to 10% serum
  • When prepared aseptically, the CyQUANT Direct 2X detection reagent is generally stable for up to 24 hours at room temperature

Table 1. PrestoBlue reagent recommended volumes.

PrestoBlue Cell Viability Reagent is supplied as a 10X solution. Bring PrestoBlue Reagent to room temperature and add directly to cells in culture medium. See table below for example volumes. PrestoBlue signal is stable for 7 hours.

Format Volume of cells + medium Volume of PrestoBlue reagent
96-well plate 90 μL 10 μL
384-well plate 36 μL 4 μL
1,536-well plate 5 μL 3 μL

Making 2X CyQUANT Direct Detection Reagent solution

1. Combine the following in a 15 mL tube:

  • Hank’s buffered saline solution, PBS, or cell culture medium—11.7 mL
  • CyQUANT Direct nucleic acid stain—48 µL
  • CyQUANT Direct background suppressor I—240 µL

2. Mix well and let stand at room temperature until ready for use. See below for example volumes. CyQUANT Direct signal is stable for 7 hours

Notes on data analysis for orthogonal assays

  1. Subtract background reading from all sample well results
  2. Use a nonlinear regression analysis of log(inhibitor) vs. response to determine EC50/IC50 value
  3. If raw response values are used, plot on dual y-axes
  4. If normalized response values are used, plot on the same axis

Differences in the EC50/IC50 determinations between PrestoBlue and CyQUANT Direct reagents reflect differences in the assay mechanism. Such orthogonal results may provide insight into the mode of action of a compound.